Introduction

In connection with the demographic situation in our country, special attention is being paid to the problem of reproductive health. According to the WHO, about 15% of married couples are infertile, and the effectiveness of their assisted reproductive technology (ART) methods does not exceed 25% [1–7]. The most common causes of female infertility and unsuccessful attempts at in vitro fertilization are endometrial pathology, the morpho functional state of which is one of the key factors determining the success of implantation and the full development of the embryo in ART cycles [8,9].  Among the pathological conditions of the endometrium, a significant place is occupied by chronic endometritis (CE), the frequency of detection of which in patients with infertility, according to different authors, ranges from 12 to 68%, reaching its greatest importance in women with tubal-peritoneal form of infertility [10–15].

Despite the enormous amount of knowledge currently accumulated on the development of pathological processes in the endometrium, there is still no single concept of the pathogenesis of CE, not only explaining the phased formation of this pathology, but also revealing the pathogenetic basis of reproductive dysfunction on its background. It is well known that chronic inflammation in the endometrium at the initial stages of its development is supported by a constant persistence of the microbial agent, which in itself is caused by pathogenic characteristics of the microorganism, on the one hand, and various defects in the microorganism’s immune defence system, on the other [16,17].

A reflection of the latter is the development of a local immunodeficiency state with the deposition of pathogenic immune complexes on the walls of the glands and blood vessels of the endometrium, which, in turn, due to the activation of the compliment system and other inflammatory mediators, contributes to the maintenance of the inflammatory process in the endometrium. In the future, with a prolonged course of CE, the immunological changes occurring in the endometrium (pathological lymphohistioplasmocytic infiltration of the stroma, changes in the phenotypic composition of cells and the ratio of Th1- and Th2-type cytokines) themselves begin to act as an etiological factor in the violation of implantation processes and the development of chorion, with subsequent formation of women infertility autoimmune genesis [18,19].

In this regard, in-depth data in the study of immune changes will make it possible to develop new methods for the diagnosis and treatment of this pathology, and reduce the level of chronicity of the process, and the economic costs in the treatment of this pathology [20–22].

 Material and methods

Under constant supervision in the gynaecological department of the Kursk City Maternity Hospital there were 30 patients of reproductive age (18-35 years old). Patients were included in the study on the basis of informed consent.  All patients underwent a comprehensive clinical and instrumental examination according to generally accepted standards, and in all cases, there was a verification of the diagnosis of chronic endometritis in the stage of incomplete remission. Women were divided into two equal groups, randomized by age, minimal concomitant diseases in remission, predicted disease severity, those who all underwent endoscopic surgery for infertility. Patients of the first group received traditional treatment, antibacterial, taking into account the sensitivity of the pathogen, antimycotic, antiviral, non-steroidal anti-inflammatory drugs, probiotics, funds aimed at restoring the vaginal microflora.  The second part of the patients received an additional course of intravenous laser blood irradiation (VLOK) using the Mulat laser therapy apparatus (Russia) with a wavelength of 0.63 microns and an output power of 2 mW at the end of the OS-2 (KIVL-01) single-use optical fibre. Laser irradiation was carried out for 25 minutes in a continuous mode of radiation; the course of treatment was 7 daily procedures.

Laboratory examination was carried out immediately upon admission to the hospital and upon discharge on the 10th day.  Levels of TNF, IL-1β, IL-8, IL-18, IL-4, IL-10, IFγ, G-CSF, complement components (C₃, C₄, C₅, C_5a), C₁ inhibitor, immunoglobulins of classes M, G, A (IgM, G, A), sIgA were determined in blood plasma and vaginal cervical flush using reagent kits of Vector-Best CJSC, NPO Tsitokin, Protein Contour LLC, St. Petersburg by enzyme-linked immunosorbent assay analysis. Before and after complex treatment, red blood cells were obtained from 10 ml of heparinized blood, for which it was defended twice in 10 mm Na-phosphate buffer (pH = 7.4), containing 0.9% sodium chloride and 3% T-500 dextran, within 30 minutes at a temperature of 37ºС.  After this, the blood was centrifuged, after separation of the supernatant, the erythrocyte mass was subjected to additional purification on a chromatographic column through HBS cellulose, after which the sorption capacity of erythrocytes (SCE) and the sorption capacity of their glycocalyx (SCG) were determined [6].  Erythrocyte membranes were isolated by G.T.  Dodge membrane lipids were determined by thin layer chromatography. Protein electrophoresis was performed in the presence of sodium dodecyl sulphate in vertical plates of a polyacrylamide gel according to the method of U.K. Laemmli, proteins stained Kumashi with blue R-250.

The intensity of lipid peroxidation (LPO) was evaluated by the content of acyl hydroperoxides (AHP) and malondialdehyde (MDA) in erythrocytes formed with a coloured complex of thiabarbituric acid [23,24]. The determination of MDA and AHP was carried out using the TBK-Agat kit (Agat-Med Russia) using the Apel-330 spectrometer (Japan) at a wavelength of 535 nm and 570 nm. To assess the state of the antioxidant system, we focused on direct / competitive enzyme-linked immunosorbent assay (ELISA) with the detection of reaction products in the wavelength range 405-630 using ready-made commercial kits: Bender Med systems superoxide dismutase (SOD) activity (Austria) and Cayman Chemical catalase “(USA).  Total antioxidant activity (OAA) was determined by a method based on the degree of inhibition of ascorbate and ferro induced oxidation of Tween-80 to MDA.  The concentration of stable nitric oxide metabolites (CM_ON) was detected in the Griss reaction at a wavelength of 540 nm using an ELISA kit from R&D (England).  All ELISA results were recorded using a Sunrise microplate photometer, Tecan (Austria).

 Result and Dissection

Statistical processing of the research results was carried out according to generally accepted criteria of variational-statistical analysis with calculation of average values ​​(M) and arithmetic mean errors (m) using the Microsoft Excel 2010 software package. The significance of differences was evaluated by the U-criterion, and the relationships were established based on the coefficient Spearman’s rank correlation.  Differences with p

Upon admission to the clinic, patients showed an increase in plasma levels of pro-inflammatory cytokines (TNF by 29.4 times, IL-1β by 4.3 times, IL-8 by 8.6 times, IL-18 by 1.7 times), compensatory anti-inflammatory (IL-4 by 8.8 times, IL-10 by 6.2 times), IFγ by 2.2 times and G-CSF by 1.5 times).  Before treatment, in this category of patients there was an increase in the concentration of components (C₃, C₄, C₅, C_5a) and the C₁ inhibitor of the complement system.  In addition, an increase in the concentration of IgM with a decrease in immunoglobulins of classes G and A was detected.

At the local level, an increase in the level of TNF, IL-8, C₃ and C₄ was also established in the vaginal-cervical flush, but, unlike the system level, a decrease in IL-4, IL-10 and C₁-inhibitor of the complement system was observed.  In addition, a decrease in the concentration of secretory immunoglobulin sIgA was detected.

Standard plasma treatment was partially normalized, but not to the level of healthy donors, the content of TNF, IL-1β, IL-8, IFγ, C₃, C₄, C_5a components of complement, IgM and IgA, even more increased the level of anti-inflammatory cytokines and C₁ inhibitor of the complement system.  The concentration of IL-18, G-CSF, C₅ remained unchanged, and the IgG content was higher than that of donors.

At the local level, standard treatment corrected the content of all investigated cytokines and the C₃ component of complement, significantly increased the concentration of sIgA, but did not affect the level of C₄ and C₁ inhibitor of the complement system.

The results obtained showed that the standard pharmacotherapy in this category of patients did not have an adequate corrective effect on the impaired immune status parameters, indicating the need for additional means and methods of immunorehabilitation in the postoperative period.

As for the changes occurring in red blood cells, when admitted to patients with CE, a decrease in the level of α- and β-spectrin, ankirin, anion transport protein (ATB), pallidine, dematin, glyceraldehyde-3-phosphate dehydrogenase (G-3PD) was found in the erythrocyte membrane -FD) and glutathione-S-transferase (G-S-T), an increase in the protein content of band 4.1, actin and tropomyosin with a normal level of protein of band 4.5.  The performed operative and TT normalized the representativeness of α- and β-spectrin and tropomyosin in the erythrocyte membrane was corrected, but not to the normal parameters, the content of ankyrin, pallidine, dematin and actin, did not affect the level of ATB, protein of the band 4.1, G-3-PD and G-S-T.  The use of VLOK additionally, in comparison with pharmacological therapy, normalizes the representativeness of ATB, actin, G-3-PD, G-S-T and, to an even greater extent, corrected the level of ankyrin, band 4.1 protein, pallidine and dematin.

In patients with CE, upon admission to the clinic, a decrease in the content of phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), glycerophospholipids (GPL - the sum of LPC, PC, PE, PS and PI), sphingomyelin (SM) was revealed in the erythrocyte membrane  phospholipids (PL - the sum of GPL and SM), an increase in the level of lysophosphatidylcholine (LPC), cholesterol (C), cholesterol esters (EC) and triacylglycerols (TAG), with a normal content of phosphatidylethanolamine (PE), the sum of mono- and diacylglycerol,  ) and unesterified fatty acids (NEFA). The performed TT was closer to the parameters of healthy donors LPC, PS, PI, GPL, SM and FL, but did not affect the representativeness of PC, C, EC and TAG.  The addition of pharmacological therapy of the postoperative period of VLOK additionally normalized the content of LPC, PI, SM, EC, TAG and, to an even greater extent, correcting the level of PC, FS, GPL and FL.

When studying the indicators of red blood cell metabolism before treatment, the activation of LPO processes (increase in the concentration of MDA and AHP), a decrease in antioxidant defence factors (OAA, SOD and catalase activity) were established. In addition, an increase in the level of CMON and a decrease in the sorption parameters of the erythrocyte membrane (SCG and SCE) were revealed. The pharmacotherapy did not affect the changed indicators of the antioxidant protection of erythrocytes (SOD and OAA) and corrected the remaining parameters of the metabolic activity of red blood cells in the direction of indicators of healthy donors.  Supplementation of VLOK treatment normalized the antioxidant defence parameters and sorption indices and, to an even greater extent, correcting the LPO parameters and СМ_ON level.

Thus, it can be stated that in patients with CE before surgery and TT, 87.9% of the indicators of the structural and functional properties of red blood cells were found to be altered from the values ​​of healthy donors, respectively. Conducted comprehensive treatment normalized 10.3% of the parameters changed before treatment, but not the norm, 55.2% and left unchanged 34.5% of the indicators.  The use of VLOK was significantly more effective, since it normalized 58.6%, corrected 37.9% and left unchanged only 3.5% of the parameters.

A definite proof of the integrative processes between the terms of the laboratory status is the presence of reliable correlation between them. To do this, we analysed the matrix of multiple correlation between the immune parameters and the structural and functional properties of red blood cells in chronic endometritis at the systemic and local levels before treatment, the results of which are given below (Table 1).

Table 1: The relationship between immune parameters and structural and functional properties of red blood cells in chronic endometritis at the local level